High density Nociceptin/Orphanin FQ (N/OFQ) and its receptor (NOPr) have been found in the ventrolateral periaqueductal gray (vlPAG), a main output pathway involved in the descending pain-control system. Our previous study demonstrated that the microinjection of N/OFQ into the vlPAG markedly facilitated nociceptive responses of spinal dorsal horn neurons. The aim of the present work was to further provide evidence for the supraspinal mechanisms of action for N/OFQ-mediated nociceptive facilitation by examining the effect of N/OFQ in the vlPAG on neurotransmitter release in the descending pain-control system, including the nucleus raphe magnus (NRM), nucleus reticularis gigantocellularis (NGC) and dorsal horn of the spinal cord. The results showed that the microinjection of N/OFQ into the vlPAG produced robust decreases in 5-hydroxytryptamine (5-HT, serotonin), norepinephrine (NE), and gamma-aminobutyric acid (GABA), and increase in glutamate (Glu) release in the spinal dorsal horn. Spinal application of 5-HT, 2-Me-5-HT (5-HT(3) receptor agonist), muscimol (GABA(A) receptor agonist), and baclofen (GABA(B) receptor agonist) significantly blocked intra-vlPAG-induced facilitation on nociceptive responses. However, the extracellular concentrations of these neurotransmitters in the NRM and NGC exhibited diversity following intra-vlPAG of N/OFQ. In the NRM, intra-vlPAG injection of N/OFQ significantly decreased 5-HT, NE, and Glu, but increased GABA release. Differently, in the NGC, both NE and GABA releases were attenuated by intra-vlPAG of N/OFQ, whereas the concentration of 5-HT and Glu exhibited a trend to increase. These findings provide direct support for the hypothesis that intra-PAG of N/OFQ-induced facilitation of nociceptive responses is associated with the release of 5-HT, NE, and amino acids.